Filoviruses include Ebola virus (EBOV) and Marburg virus (MARV), which can have up to 90% clinical fatality. These viruses are classified as category A pathogens by the NIH as they pose a serious public health and national security risk. Filoviruses have a filamentous lipid-envelope and despite being discovered more than 30 years ago, not much is known on how they replicate and spread from the host cell plasma membrane. Filoviruses contain a negative sense RNA genome, which encodes for seven proteins. The viral matrix protein VP40, which is one of the seven genes, regulates assembly and budding from the host cell membrane. VP40 completely underlies the viral lipid envelope, but to date, little is known about how VP40 is trafficked to the plasma membrane and interacts with human cell membranes. VP40 has recently been shown to form different protein structures throughout the viral life cycle and thus it is referred to as a transformer protein. The goal of this application s to unravel the complex mechanisms that mediate VP40 transport and lipid-dependent assembly. The central hypothesis is that VP40 dimers are sensitive to the phosphatidylserine (PS) concentration gradient along the secretory pathway, which regulates the VP40 structures that arise. Our preliminary data indicate that the PS content of biological membranes regulates VP40 membrane binding and hexamerization. VP40 dimers are able to bind membranes with PS content similar to that of the secretory vesicles, while VP40 oligomerization is sensitive to a higher PS content associated with the inner leaflet of the plasma membrane. This R21 application describes experiments to provide a biochemical map of how VP40 dimers move and transform into hexamers. Specific Aim 1 of the proposal will dissect the role of PS content in regulating VP40 structures in membrane sensing and transport. We will use several in vitro and cellular biochemical and biophysical assays to establish how these structures interact with cellular membranes. In order to understand the nature of complex lipid interactions between VP40 and lipids, we will further develop a controllable enzymatic PS depletion system to study live cell dynamics of VP40 assembly. Specific aim 2 will elucidate the role of PS and other important biophysical properties of the plasma membrane in early, mid-, and late stage VP40 assembly. Preliminary studies demonstrate VP40 oligomers are sensitive to plasma membrane fluidity and quantitative real-time dynamics of VP40 assembly and budding will be assessed to test the central hypothesis. Taken together, these studies should produce new and important mechanistic insight into how an Ebola virus particle forms from the plasma membrane of cells.